Identification of the Loci of Interaction between the E1 and E2 Subunits of the Pyruvate Dehydrogenase Complex from Escherichia coli

Abstract

Earlier, we reported that residues 1–45 of Escherichia coli pyruvate dehydrogenase E1 subunit interact with the second E2 subunit (Park et al., 2004 Biochemistry). Substitution of the highly conserved E1 residues, D7, D9, P10, I11, E12, T13, R14, and D15 by A, D or N, identified D7, D9, E12, R14, and D15 to be involved in E1–E2 interactions. We report charge-reversed substitutions at D9, E12, E21, E26, E27, E30, D37, and E42 to arginine. Also, the highly conserved residues on E2, R129 and R150 were found to bind to E1. Apparently, the positively charged residues on the subunit-binding domain of E2 interact with the negatively charged N-terminal residues on E1. While the E21R, E26R, D37R, and E42R E1 variants could form a complex with E2 and produce NADH in the overall assay, the D9R, E12R, E27R and E30R variants led to weakened interaction with PDHc-E2 in both respects. The folding ability of E1 was especially affected by the E30R substitution. According to MALDI-MS, the D9R E1 substitution did not impair its ability to reductively acetylate independently expressed lipoyl domain. Apparently, D9, E12, E27, and E30 on E1 have a role in binding E2, not in decarboxylating pyruvate. Supported by NIH GM-050380.